Molecular and Structural Characterizations of Lipases from Chlorella by Functional Genomics

Microalgae have been poorly investigated for new-lipolytic enzymes of biotechnological interest. In silico study combining analysis of sequences homologies and bioinformatic tools allowed the identification and preliminary characterization of 14 putative lipases expressed by Chlorella vulagaris. These proteins have different molecular weights, subcellular localizations, low instability index range and at least 40% of sequence identity with other microalgal lipases. Sequence comparison indicated that the catalytic triad corresponded to residues Ser, Asp and His, with the nucleophilic residue Ser positioned within the consensus GXSXG pentapeptide. 3D models were generated using different approaches and templates and demonstrated that these putative enzymes share a similar core with common α/β hydrolases fold belonging to family 3 lipases and class GX. Six lipases were predicted to have a transmembrane domain and a lysosomal acid lipase was identified. A similar mammalian enzyme plays an important role in breaking down cholesteryl esters and triglycerides and its deficiency causes serious digestive problems in human. More structural insight would provide important information on the enzyme characteristics.

Cystathionine-β-synthase: Molecular Regulation and Pharmacological Inhibition

Cystathionine-β-synthase (CBS), the first (and rate-limiting) enzyme in the transsulfuration pathway, is an important mammalian enzyme in health and disease. Its biochemical functions under physiological conditions include the metabolism of homocysteine (a cytotoxic molecule and cardiovascular risk factor) and the generation of hydrogen sulfide (H2S), a gaseous biological mediator with multiple regulatory roles in the vascular, nervous, and immune system. CBS is up-regulated in several diseases, including Down syndrome and many forms of cancer; in these conditions, the preclinical data indicate that inhibition or inactivation of CBS exerts beneficial effects. This article overviews the current information on the expression, tissue distribution, physiological roles, and biochemistry of CBS, followed by a comprehensive overview of direct and indirect approaches to inhibit the enzyme. Among the small-molecule CBS inhibitors, the review highlights the specificity and selectivity problems related to many of the commonly used “CBS inhibitors” (e.g., aminooxyacetic acid) and provides a comprehensive review of their pharmacological actions under physiological conditions and in various disease models.

Are Glucosylceramide-Related Sphingolipids Involved in the Increased Risk for Cancer in Gaucher Disease Patients? Review and Hypotheses

The roles of ceramide and its catabolites, i.e., sphingosine and sphingosine 1-phosphate, in the development of malignancies and the response to anticancer regimens have been extensively described. Moreover, an abundant literature points to the effects of glucosylceramide synthase, the mammalian enzyme that converts ceramide to β-glucosylceramide, in protecting tumor cells from chemotherapy. Much less is known about the contribution of β-glucosylceramide and its breakdown products in cancer progression. In this chapter, we first review published and personal clinical observations that report on the increased risk of developing cancers in patients affected with Gaucher disease, an inborn disorder characterized by defective lysosomal degradation of β-glucosylceramide. The previously described mechanistic links between lysosomal β-glucosylceramidase, β-glucosylceramide and/or β-glucosylphingosine, and various hallmarks of cancer are reviewed. We further show that melanoma tumor growth is facilitated in a Gaucher disease mouse model. Finally, the potential roles of the β-glucosylceramidase protein and its lipidic substrates and/or downstream products are discussed.

Inorganic polyphosphate in mammals: where's Wally?

Inorganic polyphosphate (polyP) is a ubiquitous polymer of tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. In prokaryotes and lower eukaryotes, both the presence of polyP and of the biosynthetic pathway that leads to its synthesis are well-documented. However, in mammals, polyP is more elusive. Firstly, the mammalian enzyme responsible for the synthesis of this linear biopolymer is unknown. Secondly, the low sensitivity and specificity of available polyP detection methods make it difficult to confidently ascertain polyP presence in mammalian cells, since in higher eukaryotes, polyP exists in lower amounts than in yeast or bacteria. Despite this, polyP has been given a remarkably large number of functions in mammals. In this review, we discuss some of the proposed functions of polyP in mammals, the limitations of the current detection methods and the urgent need to understand how this polymer is synthesized.

Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2-β-d-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2-β-d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9-β -d-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

Fructose 1,6-bisphosphatase: getting the message across

Fructose 1,6-bisphosphatase (FBPase) is a key enzyme in gluconeogenesis. It is a potential drug target in the treatment of type II diabetes. The protein is also associated with a rare inherited metabolic disease and some cancer cells lack FBPase activity which promotes glycolysis facilitating the Warburg effect. Thus, there is interest in both inhibiting the enzyme (for diabetes treatment) and restoring its activity (in relevant cancers). The mammalian enzyme is tetrameric, competitively inhibited by Fructose 2,6-bisphosphate and negatively allosterically regulated by AMP. This allosteric regulation requires information transmission between the AMP binding site and the active site of the enzyme. A recent paper by Topaz et al. (Bioscience Reports (2019) 39, pii:BSR20180960) has added additional detail to our understanding of this information transmission process. Two residues in the AMP binding site (Lys112 and Tyr113) were shown to be involved in initiating the message between the two sites. This tyrosine residue has recently be shown to be important with protein’s interaction with the antidiabetic drug metformin. A variant designed to increase metal ion affinity (M248D) resulted in a five-fold increase in enzymatic activity. Interestingly alterations of two residues at the subunit interfaces (Tyr164 and Met177) resulted in increased responsiveness to AMP. Overall, these findings may have implications in the design of novel FBPase inhibitors or activators.

Realtime 2-5A kinetics suggests interferons β and λ evade global arrest of translation by RNase L

Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here we developed a biosensor for 2’,5’-oligoadenylate (2-5A), the natural activator of RNase L. We found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes (ISGs) and secreted IFNs of types I and III (IFN-β and IFN-λ). Our data suggests that IFNs escape from the action of RNase L on translation. We propose that 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.SignificanceRNase L is a mammalian enzyme that can stop global protein synthesis during interferon response. Cells must balance the need to make interferons (which are proteins) with the risk to lose cell-wide translation due to RNase L. This balance can most simply be achieved if RNase L was activated late in the interferon response. However, we show by engineering a biosensor for the RNase L pathway, that on the contrary, RNase L activation precedes interferon synthesis. Further, translation of interferons evades the action of RNase L. Our data suggest that RNase L facilitates a switch of protein synthesis from homeostasis to specific needs of innate immune signaling.